GSM5282071: DND41 DMSO LEF1 ChIPseq rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
LEF1
Cell type
Cell type Class
Blood
Cell type
DND-41
Primary Tissue
Blood
Tissue Diagnosis
Leukemia
Attributes by original data submitter
Sample
source_name
DND41 cell line
cell line
DND41 cell line
cell type
T-ALL cell line
chip antibody
LEF1
drug treatment
DMSO
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP-seq for histone marks was performed as previously described (Petrovic et al., 2019). Briefly, 1 x 10^7 cells were crosslinked with 1% formaldehyde and lysed. Nuclei were extracted and resuspended in 1% SDS and sonicated using Brandson 450 sonicator with 25% amplitude, 0.5s on, 1s off for a total of 4min 30s on. Solubilized chromatin was then diluted and cleared with IgG (CST cat# 2729S) and recombinant protein G–conjugated Agarose beads (Invitrogen #15920-010) for 1 hour at 4°C. The cleared supernatant was subsequently immunoprecipitated with antibodies recognizing H3K27ac (Active Motif cat# 39133) or H3K4me1 (Abcam cat# ab8895) overnight at 4°C. Buffers in all steps above were supplemented with protease inhibitors (Roche cat# 11697498001). Antibody-chromatin complexes were captured with recombinant protein G–conjugated Agarose beads, washed with low salt wash buffer, high salt wash buffer, LiCl wash buffer and TE buffer with 50mM NaCl and eluted. Input sample was prepared by the same approach without immunoprecipitation. ChIP-seq for transcription factors was performed as previously described (Bossen et al., 2015). Briefly, Dynabeads Protein G (ThermoFisherScientific cat# 10003D) was incubated with antibodies recognizing MAML1 (D3K7B) (CST cat# 12166S), TCF1/TCF7 (C46C7) (CST cat# c2206S), LEF1 (D6J2W) (CST cat# 76010S), EBF1 (Boller et al., 2016), SMC1a (Bethyl, cat# A300-055A), YY1 (Active motif cat# 61779) and CTCF (EMD Millipore cat# 07-729) for 8-12 hours in PBS+0.5% BSA at 4°C. 4 x 10^7 cells were crosslinked with 1% formaldehyde and 1.5mM EGS (ThermoFisherScientific cat# 21565) and sonicated using Brandson 450 sonicator with 17% amplitude, 10s on, 1min off for 10 times. Lysate was then cleared by centrifuging for 5min at 16 Kg, 4°C and incubated with antibody-bound beads and 1% Triton Tx-100 (Roche cat# 10789704001) overnight at 4°C. Buffers in all steps above were supplemented with protease inhibitors (Roche cat# 11697498001). Antibody-chromatin complexes captured on beads were then separated on magnet and washed with wash buffer 1, 2, 3, LiCl wash Buffer and TE buffer and eluted. Following elution of histone mark and transcription factor samples, RNase (Roche cat# 10109169001) and Proteinase K (Invitrogen cat# 25530-049) treatments were performed and reverse crosslinked at 65°C overnight. DNA was purified with QiaQuick PCR Purification Kit (Qiagen, cat# 28106). Libraries were then prepared using the NEBNext Ultra II DNA library Prep Kit for Illumina (NEB cat# E7645S) with single (NEB cat# E7335, cat# E7710) or dual (NEB cat# E7600, cat# E7780) indexing. Two replicates were performed for each condition. Indexed libraries were validated for quality and size distribution using a TapeStation 4150 (Agilent), quantified by KAPA Library Quantification Kit (Roche cat# KK4824) and paired-end sequenced (38 bp+38 bp) on Illumina NextSeq 550.